Next-generation transcatheter aortic valve implantation.

Objective: Transcatheter aortic valve implantation (TAVI) procedures are increasing rapidly, but the durability of tissue valve and periprocedural complications are not satisfactory. Immune reaction to the galactose-α-1,3 galactose ß-1,4-N-acetylglucosamine (α-Gal) and conventional processing protocols of cardiac xenografts lead to calcification. Next-generation TAVI needs to be made with α-Gal-free xenografts by multiple anticalcification therapies to avoid immune rejection and enhance durability, and three-dimensional (3D) printing technology to improve the procedural safety. Methods: Porcine pericardia were decellularized and immunologically modified with α-galactosidase. The pericardia were treated by space filler, crosslinked with glutaraldehyde in organic solvent, and detoxified. The sheep-specific nitinol (nickel-titanium memory alloy) wire backbone was made from a 3D-printed model for ovine aortic root. After it passed the fitting test, we manufactured a self-expandable stented valve with the porcine pericardia mounted on the customized nitinol wire-based stent. After in vitro circulation using customized silicone aortic root, we performed TAVI in 9 sheep and obtained hemodynamic, radiological, immunohistopathological, and biochemical results. Results: The valve functioned well, with excellent stent fitting and good coronary flow under in vitro circulation. Sheep were sequentially scheduled to be humanely killed until 238 days after TAVI. Echocardiography and cardiac catheterization demonstrated good hemodynamic status and function of the aortic valve. The xenografts were well preserved without α-Gal immune reaction or calcification based on the immunological, radiographic, microscopic, and biochemical examinations. Conclusions: We proved preclinical safety and efficacy for next-generation α-Gal-free TAVI with multiple anticalcification therapies and 3D-printing technology. A future clinical study is warranted based on these promising preclinical results.

Anticalcification effect of a combination of decellularization, organic solvents and amino acid detoxification on glutaraldehyde-fixed xenopericardial heart valves in a large-animal long-term circulatory model.

OBJECTIVES: We aimed to investigate the effect of a combination of anticalcification treatments, which were effective for preventing calcification in a small animal experiment, on glutaraldehyde-fixed xenopericardial valves using a large-animal long-term circulatory model. METHODS: Valved conduits were made of porcine pericardium as a leaflet and bovine pericardium as a conduit and were implanted into the right ventricular outflow tract of goats under cardiopulmonary bypass. The goats were divided into study (glutaraldehyde + combined anticalcification treatment, n = 6) and control (glutaraldehyde alone, n = 9) groups. Upon euthanization at 1 year, echocardiography and cardiac catheterization were performed. Explanted tissues were microscopically examined and analysed for measuring the calcium content. RESULTS: Haemodynamic data were obtained from 3 and 2 goats in the study and control groups, respectively. All valves, except 1, which was limited in motion, were functioning well on echocardiography; pressure gradients across the right ventricular outflow tract were lower in the study group on cardiac catheterization. On gross inspection, all leaflets remained mobile without calcific deposits in the study group, while most leaflets were heavily calcified in the control group. The calcium content in the leaflets remained low (≤4 µg/mg) in the study group. Among the leaflets explanted from goats that survived longer (>3 months), the calcium concentration was higher in the control group than in the study group [15.1 µg/mg (n = 5) vs 2.7 µg/mg (n = 5), respectively; P = 0.008). CONCLUSIONS: Porcine pericardial leaflets treated with our anticalcification protocol showed better function and less calcification than those treated with glutaraldehyde alone in the pulmonary position.

Differences in xenoreactive immune response and patterns of calcification of porcine and bovine tissues in α-Gal knock-out and wild-type mouse implantation models.

OBJECTIVES: Although bioprostheses are widely used in cardiovascular surgery, their durability is limited due to degeneration. Degeneration of bioprostheses limiting its clinical use results from multiple factors, and immune reaction has been considered to be one of the most important factors. The study objectives were to compare the mechanical characteristic differences of porcine and bovine prostheses, assess the differences in immune reaction among different species and tissues as well as elucidate bioprosthetic failure patterns in α-Gal knock-out (KO) and wild-type mouse implantation models. METHODS: Six groups of different xenogeneic tissues (porcine pericardium, aortic valve, aortic wall; bovine pericardium, aortic valve and aortic wall) were implanted into the subcutaneous tissue of the wild-type mouse (n = 4) and the KO mouse (n = 4) (four xenogeneic tissue segments per each mouse). Mechanical and chemical tests, including tensile strength measurement and thermal stability test for pericardial tissues and pronase test for different xenogeneic tissues, were performed before implantation. Anti-α-Gal antibody titres (IgM and IgG antibodies) were measured using serum enzyme-linked immunosorbent assay analyses before implantation and 30, 60 and 90 days after implantation. Implanted tissues were harvested after 90 days and studied for histopathology and quantification of calcification. RESULTS: There were no significant differences in tensile strength and shrinkage temperature between the porcine and bovine pericardia, although the bovine pericardia showed a greater elasticity than the porcine pericardia (elongation at tensile strength, 74.8 ± 4.5% vs 50.0 ± 8.7%, P < 0.001). Resistance towards pronase degradation was not different among the groups of tissues (Groups 1-6, 89.1 ± 7.6, 95.1 ± 1.8, 90.3 ± 5.3, 93.7 ± 3.3, 89.1 ± 2.4 and 89.1 ± 3.0%, respectively; P = 0.061). The IgM titres of the α-Gal KO mice were significantly higher at 30 days after implantation (0.71 ± 0.27 vs 1.07 ± 0.48, P = 0.004), whereas the IgG titres of the α-Gal KO mice remained higher until 60 days after implantation (at 30 days, 0.81 ± 0.07 vs 1.28 ± 0.79, P = 0.017; at 60 days, 0.54 ± 0.16 vs 1.43 ± 1.10, P = 0.045) than those of the wild-type mice. Calcium levels of tissues implanted into the α-Gal KO mice were significantly higher than those implanted into the wild-type mice regardless of tissue type (from Groups 1-6, 4.72 ± 1.75 vs 27.76 ± 22.73 µg/mg; 3.05 ± 1.04 vs 15.90 ± 6.98 µg/mg; 2.13 ± 1.48 vs 29.76 ± 30.71 µg/mg; 1.02 ± 0.53 vs 5.97 ± 1.40 µg/mg; 3.18 ± 3.41 vs 30.55 ± 66.69 µg/mg; 6.21 ± 5.56 vs 21.65 ± 17.77 µg/mg, all P ≤ 0.002). CONCLUSIONS: Chronic immune response to the α-Gal antigen may cause more severe tissue calcification in α-Gal KO mice. Removal of α-Gal antigenicity is strongly advised in xenogeneic bioprosthetic tissue implantation.

In vivo efficacy for novel combined anticalcification treatment of glutaraldehyde-fixed cardiac xenograft using humanized mice.

The animal immune response against Galα1,3-Galß1-4GlcNAc-R(α-Gal) epitopes gives an important cause for the failure of glutaraldehyde(GA)-fixed cardiac xenografts. This study aimed to assess the in vivo effect of our novel combined anticalcification treatment, which includes immunologic modification, using α1,3-galactosyltransferase knock-out mice to mimic human immunologic environment. Bovine pericardia were cross-linked with GA and treated with decellularization, immunologic modification with α-galactosidase, space-filler with polyethylene glycol, organic solvent, and detoxification. The bovine pericardia were subcutaneously implanted into humanized and wild type mice, and titers of anti α-Gal IgM and IgG were evaluated at various time intervals. In vivo calcification and immunohistochemistry staining was assessed for the explanted xenografts several months after implantation. In humanized mice, titers for anti α-Gal IgM and IgG increased as the period of implantation increased, and reduced with our anticalcification treatments. The humanized mice had more in vivo calcification in GA-fixed xenografts treated with our anticalcification protocol compared with wild type mice. In humanized mice, in vivo calcification reduced with our combined anticalcification treatment, and the immunohistochemistry of the harvested xenografts proved the compatible findings with the results of in vivo immunogenicity and calcification. Humanized mice are effective model for the assessment of in vivo calcification, and our combined anticalcification treatments reduced in vivo calcification as well as in vivo immunogenicity in humanized mice group, suggesting that the animal immune reaction is the cause for calcification. Our novel combined anticalcification strategies of decellularization, immunologic modification, space-filler, organic solvent, and detoxification have possible promise to prolong the lifespan of cardiac xenograft.

Development of novel combined anticalcification protocols including immunologic modification for prolonged durability of cardiac xenograft: preclinical study using large-animal long-term circulatory models.

Cardiac xenografts are conventionally cross-linked with glutaraldehyde (GA) to impart tissue stability, reduce antigenicity, and maintain tissue sterility. However, GA-fixed xenografts are prone to calcification after long-term implantation in humans, because of phospholipids, free aldehyde groups, and residual antigenicity. We evaluated preclinical safety and efficacy using large-animal long-term circulatory models for our novel combined anticalcification protocol including immunological modification, which had been proven effective in small animal experiments. Bovine/porcine xenografts were treated with decellularization, immunological modification with α-galactosidase, GA fixation with organic solvent, and detoxification with glycine. Valve conduits made of these xenografts were transplanted into the pulmonary root of goats, and hemodynamic, radiological, immunohistopathological, and biochemical results were obtained for 12 months after implantation. Evaluation of echocardiography and cardiac catheterization demonstrated good hemodynamic status and function of the pulmonary xenograft valves. Durability of the xenografts was well preserved without calcification by specimen radiography and immunohistopathological examination. The calcium concentrations of the explanted xenografts were lower than the control xenografts. This preclinical study using large-animal long-term circulatory models demonstrated that our synergistic and simultaneous employment of multiple anticalcification therapies and novel tissue treatments, including immunological modifications, have promising safety and efficacy and should be examined further in future clinical studies.

Development of a next-generation tissue valve using a glutaraldehyde-fixed porcine aortic valve treated with decellularization, α-galactosidase, space filler, organic solvent and detoxification.

OBJECTIVES: Conventional crosslinking with glutaraldehyde (GA) renders cardiac xenografts inert, non-biodegradable and non-antigenic, but is a main cause for dystrophic calcification due to phospholipids, free aldehyde groups and residual antigenicity. A significant immune reaction to the galactose-α-1,3 galactose ß-1,4-N-acetylglucosamine (α-Gal) of a GA-fixed cardiac xenograft occurs, leading to calcification. We developed a next-generation α-Gal-free tissue valve with GA-fixed cardiac xenografts, treated using a novel combined anticalcification protocol including immunological modification, which was demonstrated effective in a small animal study. METHODS: Porcine aortic valves were decellularized with 1% sodium dodecyl sulphate, 1% Triton X-100 and 1% sodium lauroyl sarcosinate and immunologically modified with α-galactosidase. The valves were treated by a polyethylene glycol space filler, fixed with GA in 75% ethanol + 5% octanol and detoxified with glycine. We manufactured the tissue valve with the porcine aortic valve mounted on a Nitinol (nickel-titanium memory alloy) plate. The tissue valve was placed under in vitro mock circulation, and durability from mechanical stress was evaluated for 100 days. Ten sheep underwent mitral valve replacement with the tissue valve, and haemodynamic, radiological, immunohistopathological and biochemical results were obtained for 18 months after implantation. RESULTS: The in vitro circulation experiment demonstrated that the valve functioned well with good morphology. Eight sheep survived for 1, 2, 5, 10, 14, 53, 546 and 552 days after mitral valve replacement, but two sheep did not survive. An evaluation by echocardiography and cardiac catheterization demonstrated good haemodynamic status and function of the mitral valve at 18 months after implantation. The xenografts were well preserved without a α-Gal immune reaction or calcification based on the immunological, radiographic, microscopic and biochemical examinations. CONCLUSIONS: We developed a next-generation α-Gal-free tissue valve with simultaneous use of multiple anticalcification therapies and novel tissue treatments such as decellularization, immunological modification with α-galactosidase, space filler, an organic solvent and detoxification. Future investigations should evaluate α-Gal-free substitutes such as our tissue valve, and a future clinical study is warranted based on these promising preclinical results.

Valved conduit with glutaraldehyde-fixed bovine pericardium treated by anticalcification protocol.

BACKGROUND: A preclinical study was conducted for evaluating a valved conduit manufactured with a glutaraldehyde (GA)-fixed bovine pericardium treated using an anticalcification protocol. METHODS: Bovine pericardia were decellularized, fixed with GA in an organic solvent, and detoxified. We prepared a valved conduit using these bovine pericardia and a specially designed mold. The valved conduit was placed under in vitro circulation by using a mock circulation model, and the durability under mechanical stress was evaluated for 2 months. The valved conduit was implanted into the right ventricular outflow tract of a goat, and the hemodynamic, radiologic, histopathologic, and biochemical results were obtained for 6 months after the implantation. RESULTS: The in vitro mock circulation demonstrated that valve motion was good and that the valved conduit had good gross and microscopic findings. The evaluation of echocardiography and cardiac catheterization demonstrated the good hemodynamic status and function of the pulmonary xenograft valve 6 months after the implantation. According to specimen radiography and a histopathologic examination, the durability of the xenografts was well preserved without calcification at 6 months after the implantation. The calcium and inorganic phosphorus concentrations of the explanted xenografts were low at 6 months after the implantation. CONCLUSION: This study demonstrated that our synergistic employment of multiple anticalcification therapies has promising safety and efficacy in the future clinical study.

Novel self-expandable, stent-based transcatheter pulmonic valve: a preclinical animal study.

BACKGROUND: Because transcatheter implantation of pulmonary valve is indicated for limited-size dysfunctional right ventricular outflow tract only as a balloon-expandable stent, we investigated the feasibility of a large-diameter self-expandable valved stent and the durability of the valve after >6 months. METHODS: We made a nitinol-wire-based, self-expandable valved stent with leaflets made from porcine pericardium. The porcine pericardium was treated with α-galactosidase, glutaraldehyde, and glycine after decellularization. After cutting the inguinal or cervical area, we implanted a valved stent in 12 sheep through the femoral or jugular vein by using an 18-Fr delivery catheter, controlling the catheter handles and hook block under fluoroscopic and echocardiographic guidance. RESULTS: The mean body weight of sheep was 43.9 kg. We successfully implanted valved stents (diameter: 24 mm in 7 sheep, 26 mm in 5 sheep) in good position in 8 sheep, in the main pulmonary artery (PA) in 2 sheep, and in the right ventricular outlet tract (RVOT) in 2 sheep. We sacrificed 8 sheep (6 sheep in good position, 1 sheep in the main PA, and 1 sheep in the RVOT) after >6 months. Five of the 6 sheep implanted in good position showed well-preserved valve morphology at the time of sacrifice. Histologic findings after routine sacrifice showed well-maintained collagen wave structure and no visible calcification in all explanted valve leaflets. CONCLUSIONS: Transcatheter implantation of a nitinol-wire-based, self-expandable valved stent in the pulmonic valve was feasible, and stents implanted in good position showed well-preserved valve leaflets with functional competence in the mid-term results.

The effect of space fillers in the cross-linking processes of bioprosthesis.

Glutaraldehyde (GA) is largely used in the cross-linking of collagen matrices to improve their mechanical and biological properties for applications in cardiovascular surgery. However, GA has major drawbacks, including graft degeneration, calcification, and durability. The aim of this study was to test the hypothesis that filling the interstitial space in the bovine pericardium with various space fillers could prevent tissue calcification. GA, genipin, and 1-ethyl-3(-3 dimethyl aminopropyl) carbodiimide hydrochloride fixation with spacefiller treatment have been studied in order to improve the properties of heart valve xenografts. Crosslinking efficiency of GA treated group was better than genipin or 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide/N-hydroxysuccinimide treated group in vitro mechanical, enzymatic degradation resistance tests. Space-filling samples have shown significantly reduced calcification in the rabbit intramuscular implantation model. Regardless of the filling effect, the level of calcification and the cytotoxicity was low in a genipin-treated group compared to levels in the GA-treated group. The results indicated that GA and genipin fixation with space-filler treatment were effective in anticalcification for biological tissue preservation.

In vitro toxicity of serum protein-adsorbed citrate-reduced gold nanoparticles in human lung adenocarcinoma cells.

We examined the cytotoxicity effect of the serum protein coated gold nanoparticles (AuNPs) in the A549 cells. Negatively charged AuNPs were prepared by chemical reduction using citrate. The dimension and surface charge of AuNPs were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential measurements. The AuNPs modified by the citrate anion were presumed to adsorb the serum proteins as indicated from the visible absorption spectroscopy, DLS, and quartz crystal microbalance (QCM) data. The QCM results indicated that among the constituents, fetal bovine serum (FBS) should be the major adsorbate species on the AuNPs incubated in the RPMI medium. The internalization of AuNPs into the A549 cells was also monitored using TEM and dark-field microscopy (DFM). Both methylthiazol tetrazolium (MTT) and lactate dehydrogenase (LDH) assays revealed that AuNPs were toxic as determined by their half-maximal inhibitory concentration. A flow cytometric and real-time PCR analysis of apoptotic genes along with the ATP depletion measurements suggested that AuNPs induce cell damages through extrinsic and intrinsic apoptotic pathways.